Prognosis and treatment of breast cancer

ABSTRACT

The present invention relates to an antibody which specifically binds a Spot 14 (S14 or THRSP) protein in human breast cancer cells and a method for using the same to predict disease-free survival and select treatment modalities for breast cancer. The present invention is also a method for inducing apoptosis in breast cancer cells by inhibiting the expression or activity of Spot 14. Compositions and methods for treating breast cancer are also provided.

INTRODUCTION

This patent application is a continuation of U.S. patent applicationSer. No. 13/010,258, filed Jan. 20, 2011, which is a continuation ofU.S. patent application Ser. No. 11/912,413 filed Oct. 24, 2007, issuedas U.S. Pat. No. 7,906,294, which is the national phase ofPCT/US2006/018527 filed May 12, 2006, which claims benefit of U.S.Provisional Patent Application Ser. No. 60/737,223, filed Nov. 16, 2005,and U.S. Provisional Patent Application Ser. No. 60/681,683, filed May17, 2005, the contents of each of which are herein incorporated byreference in their entireties.

This invention was made with government support under Grant No. RO1DK58961-01A2 awarded by the National Institutes of Health. The U.S.government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Spot 14, also known as thyroid hormone-responsive Spot 14 (THRSP) orS14, is a primarily nuclear protein that is abundant in tissues activein long chain fatty acid synthesis, including lactating mammary gland(Cunningham, et al. (1998) Thyroid 8:815-825). It has been shown thatthe S14 gene is located on chromosome 11q13 and is overexpressed in mostbreast cancers (Moncur, et al. (1998) Proc. Natl. Acad. Sci. USA95:6989-6994). Concordant overexpression of S14 and acetylCoA-carboxylase, the rate-determining enzyme of long-chain fatty acidsynthesis, indicates that S14 is a component of the lipogenic phenotypeobserved in aggressive breast cancers.

The lipogenic tumor phenotype is characterized by high rates of fattyacid synthesis, elevated tumor content of lipogenic enzymes such asfatty acid synthase (FAS), and dependence on lipogenesis for tumor cellgrowth (Kuhajda (2000) Nutrition 16:202-2). Cerulenin, a pharmacologicalinhibitor of fatty acid synthase has been shown to cause apoptosis ofbreast cancer cells (Pizer, et al. (1996) Cancer Res. 56:2745-2747), andinhibit the growth of human ovarian tumor cell xenografts in nude mice(Pizer, et al. (1996) Cancer Res. 56:1189-1193). Likewise, theanti-obesity drug Orlistat, also a FAS inhibitor, causes apoptosis oflipogenic prostate cancer cells in culture and in xenografts inimmunodeficient mice (Kridel, et al. (2004) Cancer Res. 64:2070-2075).

In hepatocytes, S14 and lipogenic enzymes are inducible by insulin,glucose metabolism, and thyroid hormone (Cunningham, et al. (1998)supra). The lipogenic effects of insulin are substantially mediated atthe gene level by sterol response element-binding protein 1c (SREBP-1c),a transcription factor that resides in the endoplasmic reticulum untilinsulin activates its translocation to the Golgi, where the activefragment is released by proteolysis, permitting transit to the nucleusto activate gene transcription (Foretz, et al. (1999) Proc. Natl. Acad.Sci. USA 96:12737-12742; Rawson (2003) Nat. Rev. Mol. Cell Biol.4:631-640). As in the liver, SREBP-1c may be the major driver oflipogenic gene expression. This issue has been addressed in studies ofbreast cancer specimens (Yang, et al. (2003) Exp. Cell Res.282:132-137), colon cancer specimens and cells (Li, et al. (2000) Exp.Cell. Res. 261:159-165), and prostate cancer cells (Swinnen, et al.(1997) Proc. Natl. Acad. Sci. USA 94:12975-12980; Swinnen, et al. (2000)Oncogene 19:5173-5181; Heemers, et al. (2001) Mol. Endocrinol.15:1817-1828). The studies of breast and colon cancer correlatedexpression of FAS and SREBP-1c, but did not include mechanisticexperiments. Studies in prostate cancer cells, however, directlydemonstrated dependence of androgen- and growth factor-inducedexpression of FAS on SREBP-1c. Moreover, processing of the extranuclearSREBP-1c precursor was increased by androgen induction of SREBPcleavage-activating protein (SCAP), the protein responsible forescorting SREBP-1c to the Golgi, where proteolytic activation occurs. Incontrast to the enhancement of SREBP-1c processing by androgen inprostate cancer cells, however, no increase was observed in nuclearSREBP-1c content in progestin-treated T47D cells demonstrating S14 geneinduction (Heemers, et al. (2000) Biochem. Biophys. Res. Comm.269:209-212).

A number of other proteins have been identified which may accelerate thegrowth of breast cancer cells. Such proteins include p53, atranscriptional regulator with tumor suppressor properties, nm23, aputative metastasis suppressor, and several families of cell surfacegrowth factor receptors and their cognate ligands, including theepidermal growth factor (EGF) receptor superfamily, the insulin-likegrowth factor (IGF-1) family, and the fibroblast growth factor (FGF)family. For example, HER2, a receptor with close similarity toEGF-Receptor, also known as c-erBb-2 (Coussens, et al. (1985) Science230:1132-1139; Yamamoto, et al. (1986) Nature 319:230-234; King, et al.(1985) Nature 307:521-527) has been identified. This receptor was alsoisolated as the rat oncogene neu, an oncogene responsible forchemically-induced rat glioblastomas (Padhy, et al. (1982) Cell28:865-871; Schechter, et al. (1984) Nature 312:513-516; Bargmann, etal. (1986) Nature 319:226-230). HER2/erbB-2 is known to be amplified andoverexpressed in about 25% of human breast cancers (Slamon, et al.(1987) Science 235:177-182; Slamon, et al. (1989) Science 244:707-712).

SUMMARY OF THE INVENTION

The present invention is a method of predicting disease-free survival ofa subject with breast cancer. The method involves detecting theexpression level of S14 in a sample obtained from a subject with breastcancer, comparing the level in the sample with a control, wherein a lackof overexpression of S14 in the sample compared to the control ispredictive of prolonged disease-free survival.

The present invention is also a method for selecting a therapeuticmodality for the treatment of breast cancer. The method involvesdetecting the expression level of S14 in a sample obtained from asubject with breast cancer, comparing the level in the sample with acontrol, and selecting a therapeutic modality based upon the expressionlevel of S14 in the sample.

The present invention is further an isolated antibody, antibodyfragment, or antibody derivative which is selected for specificallybinding to an S14 protein of SEQ ID NO:1 in breast tissue or cells. Anisolated hybridoma for producing the antibody and a kit containing theantibody, antibody fragment, or antibody derivative are also provided.

A method for inducing apoptosis in a breast cancer cell is alsoprovided. This method involves contacting a breast cancer cell with anagent that inhibits the expression or activity of Spot 14 therebyinducing apoptosis.

The present invention is also a method for treating breast cancer byadministering to a subject with breast cancer an effective amount of anagent that inhibits the expression or activity of Spot 14 so thatapoptosis in breast cancer cells in the subject is induced and thebreast cancer is treated.

A composition composed of an agent that inhibits the expression oractivity of Spot 14 and a pharmaceutically acceptable carrier for thetreatment of breast cancer is also provided.

The present invention is further a method for identifying an agent thatinduces apoptosis in a breast cancer cell. The method involves the stepsof contacting a breast cancer cell with a test agent and determining theexpression or activity of Spot 14 in the breast cancer cell, wherein adecrease in the expression or activity of Spot 14 in the breast cancercell contacted with the test agent, compared to a breast cancer cell notcontacted with the test agent, is indicative of an agent that inducesapoptosis in a breast cancer cell.

The present invention is also a method for identifying an agent thatinduces apoptosis in a breast cancer cell by contacting Spot 14 with atest agent and determining the multimerization of Spot 14, wherein adecrease in the multimerization of Spot 14 contacted with the testagent, compared to multimerization of Spot 14 not contacted with thetest agent, is indicative of an agent that induces apoptosis in a breastcancer cell. Agents identified by the screening methods of the inventionare also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relationship of S14 content to tumor grade and size.The frequency of strong staining in normal mammary tissue is indicatedby the bar on the y-axis. FIG. 1A, analysis of S14 staining in DCIS(n=44) as a function of histological grade. FIG. 1B, pooled data frominvasive breast cancers without (n=34) or with (n=54) lymph nodemetastases at the time of diagnosis, stratified by tumor grade. FIG. 1C,pooled data from invasive cases (n=88) as a function of tumor size.

FIG. 2 shows the disease-free survival of subjects after treatment ofinvasive breast cancer as a function of S14 expression in the primarytumor. Subjects with and without positive lymph nodes at initial surgerywere pooled for Kaplan-Meier analysis. Those with an immunohistochemicalscore of 0 or 1+ (n=21) are represented by the solid line, and thosewith a 2+ S14 score by the dotted line (n=67). The difference betweenthe two plots was statistically significant (p=0.015).

FIG. 3 shows disease-free survival in subjects with invasive breastcancer as a function of S14 scores and the presence or absence of lymphnode metastases at initial surgery. The same subjects represented inFIG. 2 were analyzed by Kaplan-Meier analysis. Subjects with negativelymph nodes and submaximal S14 scores (n=11) or positive lymph nodes andlow S14 scores (n=10) are represented by the top tracings, which aresuperimposed. There were no recurrences in those groups. Among subjectswith negative lymph nodes and a high S14 scores (n=23), one recurredafter ˜2000 days follow-up (dashed line). Among the subjects with nodalmetastasis and strong S14 staining (n=44), there were 14 recurrences(p<0.0001).

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that S14 is a marker for aggressive breastcancers. Moreover, S14 expression has been demonstrated as beingrequired for the growth and survival of breast cancer cells, asinhibiting the expression of S14 was found to induce apoptosis.Accordingly, in addition to being useful as a prognostic marker, S14 isalso useful as a target for therapeutic agents in the treatment ofbreast cancer.

Increased levels of S14 expression in tumors strongly correlated withtumor aggressiveness. The frequency of maximal S14 expression exhibiteda strong positive correlation with tumor grade in both in situ andinvasive cases, and was also associated with larger tumor size. Further,S14 overexpression was not acquired during tumor progression and the S14score did not co-segregate with that of the conventional tumor markers(sex steroid receptors, Her2/neu) or cyclin D1. Accordingly, the presentinvention relates to the use of S14 as a marker for predicting orprognosticating disease-free survival of a subject with breast cancer,or alternatively predicting cancer-attributable death, includingrecurrence, of breast cancer in a subject. By detecting the level ofexpression of S14 in a sample from the subject and comparing theexpression of S14 in the sample with the expression of S14 in a control,it can be predicted whether the subject has a non-aggressive form ofcancer and therefore, with treatment, an increase in the number of yearsof disease-free survival.

In carrying out the methods of the present invention, the level ofexpression of S14 protein in a sample can be detected using any one of avariety of immunoassay methods with qualitative or quantitative results.For a review of immunological and immunoassay procedures in general, seeStites and Terr, ed. (1991) Basic and Clinical Immunology 7th Edition.Moreover, the immunoassays of the present invention can be performed inany of several configurations, which are reviewed extensively in EnzymeImmunoassay ((1980) Maggio, ed., CRC Press, Boca Raton, Fla.) andPractice and Theory of Enzyme Immunoassays ((1985) Tijssen, ed.,Elsevier, Amsterdam, The Netherlands). It is contemplated that either anabsolute, semi-quantitative, or relative level of S14 expression can bedetected using the immunoassays disclosed herein.

While the level of S14 expression can be detected by analyzing saidlevels in breast tissue samples or aspirated breast cell samples (e.g.,a biopsy sample or is situ imaging), it is contemplated that the levelof S14 could also be detected in a bodily fluid (e.g., a blood sample ora sample of fluid obtained by breast duct lavage). In general, thesample can be obtained from a subject diagnosed with breast cancer or asubject suspected of being at risk of having breast cancer (e.g.,because of environmental factors or family history). For these subjectsthe method of the invention is useful for selecting a suitabletherapeutic course of treatment. Alternatively, the sample can beobtained from a subject that has been treated for breast cancer, whereinprognosis is desired to determine whether the subject has an aggressiveform of cancer which will recur and require more aggressive initialtherapy and closer monitoring and follow-up.

In one embodiment, an immunoassay to detect or measure S14 levels in ahuman sample uses an isolated antibody (e.g., polyclonal or monoclonal),antibody fragment, or antibody derivative which was raised to anisolated S14 protein of SEQ ID NO:1 or a fragment thereof and selectedfor specifically binding to an S14 protein in breast tissue or cells.The antibody, antibody fragment, or antibody derivative is furtherselected to have low cross-reactivity against non-target proteins andany such cross-reactivity is removed by immunoabsorption prior to use inthe immunoassay.

As used herein, an antibody refers to a polypeptide substantiallyencoded by an immunoglobulin gene or immunoglobulin genes, or fragmentsthereof which specifically bind and recognize an antigen. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit includes atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one light (about 25 kD) and oneheavy chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively. In one embodiment, the antibody of the instantinvention is polyclonal. In another embodiment, the antibody of theinstant invention is monoclonal.

For the production of polyclonal or monoclonal antibodies, eitherrecombinant S14 protein or naturally occurring S14 protein (e.g., inpure or impure form) can be used as antigen or immunogen. In particularembodiments, synthetic peptides of immunogenic portions of the S14protein (e.g., SEQ ID NO:1) are used as an antigen for the production ofantibodies to the S14 protein. Immunogenic portions of S14 can bereadily identified by the skilled artisan using any art-establishedcomputer algorithm for identifying such antigenic sequences (see, e.g.,Jamison and Wolf (1988) Bioinformatics 4:181-186; Carmenes, et al.(1989) Biochem Biophys Res Commun. 159(2):687-93).

For the production of polyclonal antibodies, various host animals can beimmunized by injection with the S14 antigen including but not limited torabbits, mice, rats, etc. Various adjuvants can be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

A monoclonal antibody, which is a substantially homogeneous populationof antibodies to a particular antigen, can be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to thehybridoma technique of Kohler and Milstein ((1975) Nature 256:495-497)and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique(Kosbor, et al. (1983) Immunology Today 4:72; Cole, et al. (1983) Proc.Natl. Acad. Sci. USA 80:2026-2030); and the EBV-hybridoma technique(Cole, et al. (1985) Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the monoclonal antibody of this invention can becultivated in vitro or in vivo. Desirably, monoclonal antibodies areproduced in vivo because of the high titers obtained.

Chimeric antibodies are also contemplated. Techniques developed for theproduction of chimeric antibodies (Morrison, et al. (1984) Proc. Natl.Acad. Sci. USA 81:6851-6855; Neuberger, et al. (1984) Nature312:604-608; Takeda, et al. (1985) Nature 314:452-454) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a murine monoclonalantibody and a human immunoglobulin constant region.

Antibodies can also be modified, e.g., to produce a number ofwell-characterized fragments generated by digestion with variouspeptidases. For example, pepsin digestion of an antibody producesF(ab)′₂. The F(ab)′₂ can further be reduced under mild conditions tobreak the disulfide linkage in the hinge region, thereby converting theF(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially aFab with part of the hinge region (see, Fundamental Immunology, ThirdEdition (1993) W. E. Paul, ed., Raven Press, NY). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such fragments can be synthesized denovo either chemically or by utilizing recombinant DNA methodology.Accordingly, the term antibody fragment also includes fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. Thus, an antibody fragmentincludes, but is not limited to, single chain antibodies, Fab fragments,F(ab′)₂ fragments, diabodies (Holliger, et al. (1993) Proc. Natl. Acad.Sci. USA 90:6444; Poljak (1994) Structure 2:1121-1123), fragmentsproduced by a Fab expression library (Huse, et al. (1989) Science246:1275-1281), and epitope-binding fragments of any of the above.

Antibody derivatives such as peptide aptamers, which are selected forspecifically binding to an S14 protein in breast tissue or cells, arealso provided in the instant invention. Peptide aptamers can berationally designed or screened for in a library of aptamers (e.g.,provided by Aptanomics SA, Lyon, France). In general, peptide aptamersare synthetic recognition molecules whose design is based on thestructure of antibodies. Peptide aptamers consist of a variable peptideloop attached at both ends to a protein scaffold. This double structuralconstraint greatly increases the binding affinity of the peptide aptamerto levels comparable to that of an antibody (nanomolar range).

The phrase “specifically (or selectively) binding to an S14 protein inbreast tissue or cells” refers to a binding reaction which isdeterminative of the presence of the S14 protein in its native,three-dimensional conformation in the presence of a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to S14 protein anddo not bind in a significant amount to other proteins present in thesample. Specific binding of an antibody under such conditions mayrequire an antibody that is selected for its specificity for S14protein. For example, antibodies raised to an S14 protein fragment ofSEQ ID NO:1 can be selected to obtain antibodies specificallyimmunoreactive with S14 protein in its native conformation in breasttissue or breast cells and not with other proteins except forpolymorphic variants. Typically a specific or selective reaction will beat least twice background signal or noise and more typically more than10 to 100 times background. A variety of immunoassay formats can be usedto select antibodies specifically immunoreactive with S14 in breasttissue or breast cells. For example, solid-phase ELISA immunoassays,western blot analysis, or immunohistochemistry are routinely used toselect monoclonal antibodies specifically immunoreactive with a proteinin a particular tissue or cell. See Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity. Specific monoclonal and polyclonalantibodies will usually bind with a K_(D) of at least about 0.1 mM, moreusually at least about 1 μM, or at least about 0.1 μM or better.

In particular embodiments, an isolated antibody, antibody fragment, orantibody derivative which is selected for specifically binding to an S14protein of SEQ ID NO:1 in breast tissue or cells is a component of a kitfor detecting the expression level of S14 in a sample. A kit of theinvention generally includes a container containing the isolatedantibody, antibody fragment, or antibody derivative. The kit can alsocontain other solutions necessary or convenient for detecting theinvention the expression level of S14. The container can be made ofglass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc.The kit may also contain written information, such as procedures forcarrying out the method of the present invention or analyticalinformation, such as the amount of reagent contained in the firstcontainer means and representative images of breast cancer tumor sampleswith no detectable S14 expression (e.g., semi-quantitatively graded 0),low level S14 expression (e.g., semi-quantitatively graded +1) or a highlevel of S14 expression (e.g., semi-quantitatively graded +2). Thecontainer can be in another container, e.g., a box or a bag, along withthe written information.

As indicated supra, any one of a variety of immunoassay methods can beused to detect and measure the level of expression of S14 protein in asample. Typically, such immunoassays often utilize a labeling agent tospecifically bind to and label the binding complex formed by theantibody and the antigen. The labeling agent can itself be one of thecomponents of the antibody/antigen complex. Thus, the labeling agent canbe a labeled S14 protein or a labeled anti-S14 protein antibody.Alternatively, the labeling agent can be a third moiety, such as anotherantibody (i.e., a secondary antibody), that specifically binds to theantibody/S14 protein complex. Alternatively, the secondary antibody maylack a label and be bound by a labeled tertiary antibody specific toantibodies of the species from which the secondary antibody is derived.The secondary antibody can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

The particular labeling agent or detectable group used in the assay isnot a critical aspect of the invention, so long as it does notsignificantly interfere with the specific binding of the antibody usedin the assay. The detectable group can be any material having adetectable physical or chemical property. Such detectable labels havebeen well-developed in the field of immunoassays and, in general, mostany label useful in such methods can be applied to the presentinvention. Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluoresceinisothiocyanate, TEXAS RED™, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc.) beads.

The label can be coupled directly or indirectly to the desired componentof the assay according to methods well-known in the art. As indicatedabove, a wide variety of labels can be used, with the choice of labeldepending on sensitivity required, ease of conjugation with the assaycomponent or moiety, stability requirements, available instrumentation,and disposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the assaycomponent or moiety of interest. The ligand then binds to an anti-ligand(e.g., streptavidin) molecule which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. A number ofligands and anti-ligands can be used. Thyroxine, and cortisol can beused in conjunction with the labeled, naturally occurring anti-ligands.Alternatively, any haptenic or antigenic compound can be used incombination with an antibody.

The components of the assay can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904).

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G can also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol. 111:1401-1406 andAkerstrom, et al. (1985) J. Immunol. 135:2589-2542).

Immunoassays for detecting S14 protein in samples can be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of S14 protein is directly measured. In one type ofsandwich assay, for example, the anti-S14 protein antibodies can bebound directly to a solid substrate where they are immobilized. Theseimmobilized antibodies then capture S14 protein present in the testsample. S14 protein thus immobilized is then bound by a labeling agent,such as a second S14 protein antibody bearing a label. Alternatively,the second antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second can be modified with adetectable moiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

In competitive assays, the amount of S14 protein present in the sampleis measured indirectly by measuring the amount of an added (exogenous)S14 protein displaced (or competed away) from an anti-S14 proteinantibody by the S14 protein present in the sample. In one competitiveassay, a known amount of, in this case, the S14 protein is added to thesample and the sample is then contacted with an anti-S14 antibody. Theamount of S14 protein bound to the antibody is inversely proportional tothe concentration of S14 protein present in the sample. In oneembodiment, the antibody is immobilized on a solid substrate. The amountof the S14 protein bound to the antibody can be determined either bymeasuring the amount of S14 protein present in a S14 protein/antibodycomplex, or alternatively by measuring the amount of remaininguncomplexed protein.

A hapten inhibition assay is another competitive assay. In this assayS14 protein is immobilized on a solid substrate. A known amount ofanti-S14 protein antibody is added to the sample, and the sample is thencontacted with the immobilized S14 protein. In this case, the amount ofanti-S14 protein antibody bound to the immobilized S14 protein isinversely proportional to the amount of S14 protein present in thesample. Again, the amount of immobilized antibody can be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection can be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody.

As an alternative to competitive and noncompetitive assays, western blot(immunoblot) analysis can be used to detect and quantify the presence ofS14 protein in the sample. The technique generally encompassesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind S14 protein. These antibodies can be directlylabeled or alternatively can be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-S14 protein antibodies.

Immunohistochemical analysis can also be conducted tosemi-quantitatively detect the levels of S14 in a tissue sample comparedto a control. In general, tissue section samples are overlaid with ablocking solution and subsequently contacted with an anti-S14 antibody.The sample sections are generally overlaid with the antibody solutionfor 10-20 hours and subsequently washed to remove unbound antibody. TheS14 protein/antibody complex is then detected either directly orindirectly as described above. Immunohistochemical techniques arewell-known to those of skill in the art (see, e.g., “Methods inImmunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons,1980; Campbell et al., “Methods and Immunology”, W. A. Benjamin, Inc.,1964; and Oellerich, M. (1984) J. Clin. Chem. Clin. Biochem.22:895-904).

Throughout the assays disclosed herein, incubation and/or washing stepsmay be required after each combination of reagents. Incubation steps canvary from about 5 seconds to several hours, preferably from about 5minutes to about 24 hours. However, the incubation time will depend uponthe assay format, antigen, volume of solution, concentrations, and thelike. Usually, the assays will be carried out at ambient temperature,although they can be conducted over a range of temperatures, such as 10°C. to 40° C.

Means of detecting labels are well-known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means a microscope or by photographic film, by theuse of electronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels can bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Further, simple calorimetriclabels can be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

While detecting or measuring the level of S14 protein levels areexemplified herein, it is contemplated that the level of S14 mRNAexpression could also be used as an indicator of disease-free survival.Methods for detecting the transcript levels are well-known in the artand include, but are not limited to, RT-PCR, chip-based analysis,northern blot analysis, and the like. However, the detection of S14 mRNAin breast cancer samples is desirably carried with samples which havebeen dissected (e.g., by laser-assisted microdissection) to removetissues of adipose origin.

Upon cluster analysis, with JAVA TREEVIEW software, of unfiltered dataobtained from a 24,000-gene array of 147 cases of breast cancer(University of North Carolina Microarray Database), S14 expression wasfound to cluster with a group of 29 genes, including several that werereadily identifiable as adipocyte-specific, including perilipin,hormone-sensitive lipase, adipocyte fatty acid binding protein 4,adiponectin, and lipoprotein lipase (LPL). This data indicated that theprobe used on the arrays contained mRNA from adipocytes of the mammaryfat pad. The “co-regulation” of some genes in this cluster may thereforereflect the variable admixture of adipocyte and tumor mRNA.

To examine this further, a panel of cell lines were assessed forexpression of LPL mRNA by RT-PCR. Human pre-adipocyte and adipocyte mRNAserved as negative and positive controls, respectively. No expression ofLPL mRNA was observed in a variety of lipogenic breast cancer cell lines(ZR75.1, SKBR3, MCF7, T47D+/−progestin), or mammary epithelium (MCF10a).Hepatoma (HepG2) and embryonic kidney (HEK293) likewise did not expressit. Importantly, a cervical carcinoma line (HeLa) that expressesnegligible levels of FAS (Oskouian (2000) Cancer Letters 149:43-51), didexpress LPL mRNA. This indicates that expression of LPL may confer anadvantage to tumor cells that have a low capacity for de novolipogenesis. Overall, these observations confirmed the LPL mRNA detectedon breast cancer microarrays was of adipose origin, and indicate thatmicroarray data for genes that are expressed in both breast tumors andadipocytes, such as S14, FAS, and PPAR-γ, are not interpretable unlesstumor samples are dissected to obtain probes representative of tumortissue.

Once the level of S14 expression (protein or mRNA) in the sample isdetected or measured, it is compared to the levels of S14 expression inone or more controls in order to predict whether the subject from whichthe sample was obtained has an aggressive form of cancer and therefore adecrease in the number of years of disease-free survival or whether thesubject has a less aggressive form of cancer with a decreased risk ofrecurrence and increased long-term survival. As exemplified herein, acontrol can be a sample obtained from healthy subject (i.e., a subjectwithout any signs or symptoms of breast cancer) or can be arepresentative breast cancer tumor sample with no detectable S14expression (e.g., semi-quantitatively graded 0), low level S14expression (e.g., semi-quantitatively graded +1) or a high level of S14expression (e.g., semi-quantitatively graded +2). By using one or morecontrol samples (e.g., in a panel), the skilled pathologist can comparethe intensity of staining of S14 in an immunohistochemical assay for asemi-quantitive, or absolute level of expression of S14 in an ELISA orRT-PCR for a quantitative determination of S14 levels in a test samplefrom a subject. As exemplified herein, a sample is said to lack S14overexpression when S14 levels in the sample are comparable to S14levels in samples from healthy subjects or breast cancer samples graded0 or +1. In contrast, a sample is said to overexpress S14 when S14levels in the sample are comparable to S14 levels in breast cancersamples graded +2. In accordance with the method of the invention,subjects lacking S14 overexpression will generally survive, followingsurgical removal or the primary tumor and/or chemotherapy, for a certainperiod of time without cancer recurrence (i.e., prolonged disease-freesurvival), whereas subjects with S14 overexpression generally have amore aggressive form of cancer and therefore an increased risk of cancerrecurrence with treatment. As used herein, prolonged disease-freesurvival is intended to mean disease-free survival (i.e., no breastcancer recurrence) for generally more than 5 years after treatment, ormore specifically more than 8 years after treatment, or more desirablyat least 10 years after treatment.

The predictive method of the present invention can be used clinically tomake treatment decisions by choosing the most appropriate treatmentmodalities for any particular subject. The predictive methods of thepresent invention are valuable tools in predicting if a subject islikely to respond favorably to a treatment regimen, such as surgicalintervention, chemotherapy with a given drug or drug combination, and/orradiation therapy, or whether long-term survival of the subject,following surgery and/or termination of chemotherapy or other treatmentmodalities is likely. Accordingly, the present invention is also amethod for selecting a therapeutic modality for the treatment of breastcancer. The method involves detecting the expression level of S14 in asample obtained from a subject with breast cancer, comparing the levelin the sample with a control, and based upon the compared level,selecting an appropriate therapeutic modality for the treatment of thebreast cancer in the subject. For example, a subject with S14 levelscomparable to S14 levels in breast cancer samples graded +2 may requirea more aggressive treatment modality (e.g., breast cancer surgery withchemotherapy or radiation therapy), whereas a subject lacking S14overexpression may require a less aggressive treatment modality (e.g.,breast-conserving lumpectomy).

Having demonstrated that levels of S14 expression are indicative ofdisease-free survival, it was further determined whether S14 plays arole in breast cancer growth and survival. As SREBP-1 is a transcriptionfactor that induces the expression of genes concerned with lipogenesis(Rawson (2003) supra), the expression of SREBP-1 in cancer cells wasdetermined by RT-PCR analysis of hepatoma (HepG2, known to express bothSREBP-1 isoforms (Shimomura, et al. (1997) J. Clin. Invest.99:838-845)), lipogenic breast cancer (T47D (Chalbos, et al. (1987) J.Biol. Chem. 262:9923-9926)), and non-lipogenic cervical adenocarcinoma(HeLa (Oskouian (2000) Cancer Lett. 149:43-51)). This analysisdemonstrated that both the SREBP-1a and SREBP-1c isoforms were expressedin these cell types.

It has been shown that combined androgen/progestin R1881 or progestinR5020 significantly induces S14 mRNA in T47D cells; an effect mediatedby progestin as determined by using anti-androgen CASODEX® andanti-androgen/progestin RU146 (Heemers, et al. (2000) supra). Real-timeRT-PCR, using cyclophilin as a control, showed that S14, FAS, andSREBP-1c mRNAs were significantly induced by 10 nM R1881 within 48hours. A time course using nM R5020 showed induction of S14 and FASmRNAs comparable to that observed with R1881. There was a lag timeof >10 hours between application of the hormone and the onset ofaccumulation of S14 and FAS mRNAs. This experiment demonstratedprogestin induction of SREBP-1c mRNA. As the two compounds appeared tohave comparable actions in T47D cells, subsequent studies were primarilyperformed with R1881.

Using a human S14 gene promoter fragment fused to a luciferase reporter,the mechanism underlying R1881-induced accumulation of S14 mRNA wasdetermined. The human S14 construct contained the proximal 4003 bp ofthe S14 promoter (Campbell, et al. (2003) Endocrinol. 144:5242-5248).This fragment does not contain a canonical progesterone response element(Lieberman, et al. (1993) Mol. Endocrinol. 7:515-527). Nevertheless,R1881 induced promoter activity by 4-fold. Employing a 157 bp fragmentof the human FAS gene promoter that is also devoid of progesteroneresponse elements (Swinnen, et al. (1997) supra), the effect of R1881 onFAS gene expression was examined. A 4-fold induction was observed after48 hours exposure to the hormone, and the response from a construct withthe sterol response element deleted was markedly reduced.

SREBP-1c mutants were delivered to cells via adenoviral vectors toassess the role of SREBP-1c in S14 gene activation by progestin. Cellswere grown in charcoal-stripped fetal calf serum for 48 hours andinfected with adenoviruses (multiplicity of infection=50) for 1 hour inthe same medium. R1881 (10 nM) or vehicle was added after 8 hours, andRNA was harvested 40 hours later. S14 mRNA was induced ˜8-fold in thepresence of the control adenovirus (Ad-β-gal). Basal S14 expression wasunaffected by the dominant-negative mutant (Ad-SREBP-1c-DN), whileinduction was reduced to 2.5-fold. Constitutively active SREBP-1c(Ad-SREBP1c) in the absence of R1881 caused a major induction of S14mRNA, 330-fold over the basal value seen in the presence of Ad-β-galwithout R1881, while Ad-SREBP plus R1881 superinduced, to ˜1300-foldabove the unstimulated level.

Western blot analysis showed effects on S14 protein concordant withthose of the mRNA. Cells were grown in media containing stripped fetalcalf serum for 48 hours, and then, with or without preceding adenoviraldelivery of the constitutively active SREBP-1c mutant, exposed or not to10 nM R1881 for 48 hours. No S14 was appreciable in cells culturedwithout hormone or Ad-SREBP-1c. A faint band of the appropriate size(˜16 kD) was seen after stimulation with R1881 alone, while a strongsignal appeared after exposure to Ad-SREBP-1c. As was the case for S14mRNA, application of both stimuli induced S14 protein above the levelseen with SREBP-1c alone. The adenoviral construct encoded the matureform of SREBP-1c, and thus did not require proteolytic processing. Thesuperinduction of S14 therefore could not be ascribed to enhancement ofany component of the SREBP-1c activation apparatus, such as SCAP, as wasdemonstrated for FAS gene expression in androgen-stimulated prostatecancer cells (Heemers, et al. (2001) supra). These data indicate thatSREBP-1c is required for full induction by progestin, and also indicatethe presence of an additional, SREBP-independent mechanism.

The effects of SREBP-1c and progestin on FAS expression were alsoanalyzed. FAS mRNA was induced ˜2-fold by R1881 in the presence ofAd-β-gal. Basal FAS mRNA expression was slightly reduced byAd-SREBP-1c-DN, while hormonal induction was abrogated. Ad-SREBP1c inthe absence of R1881 induced FAS mRNA content to a level comparable tothat seen after infection with Ad-β-gal in the presence of hormone, andAd-SREBP plus R1881 further increased FAS mRNA accumulation. Westernblot analysis using an anti-FAS antibody showed less marked induction ofFAS enzyme than of S14 protein, as was the case for the respectivemRNAs, but did show a clear increase in response to the combination ofSREBP-1c and progestin compared to the response to either stimulusalone.

S14-related peptide (S14-RP) is ancestral to the S14 gene, and sharesstrong homology to three domains of S14 (Wang, et al. (2004) Gene332:79-88). RT-PCR, using two different primer pairs, indicated thatS14-RP transcripts occurred in T47D cells. To determine whether ananti-S14 antibody recognized S14-RP, a full-length human S14-RP cDNA(Open Biosystems, Huntsville, Ala.) with a hemagglutinin (HA) tagappended to the amino terminus was introduced into HEK293 cells bytransient transfection, and western analysis was performed withanti-hS14 or anti-HA antibodies. The anti-HA blot demonstrated a band ofappropriate migration (˜20 kD), but no signal was observed on the blotprobed with the anti-hS14 antibody. Thus, the anti-hS14 antibody doesnot recognize S14-RP.

In hepatocytes, induction of lipogenic gene expression requires thepresence of two distinct signals, one triggered by insulin, and theother by glucose metabolism (Foretz, et al. (1999) supra). Insulinsignals through SREBP-1c, and glucose metabolism is sensed by aliver-specific carbohydrate-responsive factor termed carbohydrateresponse element-binding protein (CHREBP) (Yamashita, et al. (2001)Proc. Natl. Acad. Sci. USA 98:9116-9121). Moreover, PCG-1β may directlyfacilitate the action of SREBP-1c in hepatocytes. To determine whetherthese proteins mediate superinduction of S14 gene expression by SREBP-1cand progestin, PCG-1β and CHREBP expression in T47D cells was analyzedusing RT-PCR. No evidence of PGC-1β gene expression was observed. In thecase of CHREBP, however, a band of the expected size was amplified fromT47D cell template.

In contrast to SREBP-1 mRNA, real-time RT-PCR analysis indicated thatCHREBP mRNA was not induced by progestin. A panel of five shortinhibitory RNAs (siRNAs) were assessed for inhibition of CHREBPexpression. None were effective in reducing levels of CHREBP mRNA,including an siRNA corresponding to the human homolog (5′-tac gtc ggcaat gct gac at-3′, SEQ ID NO:2) of a mouse sequence which has beensuccessfully used to reduce levels of mouse CHREBP (5′-tat gtt ggc aatgct gac at-3′, SEQ ID NO:3) (Dentin, et al. (2004) J. Biol. Chem.279:20314-20326).

There has been an indication that progestin induces glucose transporterexpression in lipogenic ZR-75-1 human breast cancer cells, suggesting alink between sex steroids and glucose in lipogenic gene regulation inbreast cancer cells (Medina, et al. (2003) Endocrinology 144:4527-4535).While CHREBP mRNA was readily detectable in T47D breast cancer cells, nomajor induction of S14 or FAS mRNAs was observed in response toincreasing the glucose concentration in culture media from 5.5 to 27 mM,a stimulus that is sufficient for maximal induction oflipogenesis-related genes in hepatocytes (Foretz, et al. (1999) Mol.Cell. Biol. 19:3760-3768). To assess the potential role of glucosesignaling in lipogenic gene regulation in T47D cells, cells wereacclimatized for 3 days to media containing 5.5 mM glucose and strippedfetal calf serum. Subsequently some wells were switched to high-glucosemedium (27 mM) with or without 10 nM R1881 for 48 hours. Analysis of S14mRNA by real-time RT-PCR revealed only minor induction by glucose, whichin the presence of hormone was not statistically significant. Thus,glucose signaling through CHREBP does not appear to mediate the S14superinduction.

Enforced overexpression of S14 was achieved by infecting cells withadenovirus harboring a full-length rat S14 cDNA. Using an antibodyspecific for rat S14, western blot analysis of cell lysates collected 3days after adenoviral infection revealed no signal from cells infectedwith control adenovirus (Ad-β-gal), or an adenovirus harboring the S14cDNA in the antisense orientation. Infection with Ad-rS14, however,yielded a strong band of the appropriate size (˜17 kD), of intensitycomparable to that observed in liver from a hyperthyroid rat fed afat-free, high carbohydrate diet. Infection with Ad-rS14 acceleratedaccumulation of viable cells by 45% above that observed after infectionwith Ad-β-gal after 5 days. Similar effects were observed in MCF7 andSKBR3 breast cancer, but not MCF10a mammary epithelial or HepG2hepatocarcinoma cells.

To reduce S14 mRNA and protein expression, siRNA targeting S14 wereemployed. T47D cells were difficult to transfect with siRNA, owing tovariable transfection efficiency between experiments. The cause of thevariability was not clear, except that highly passaged cells appearedless susceptible to transfection. Using low passage number cells (<8),fluorescent-tagged siRNA (fl-siRNA) were transfected in each experiment.Four hours after transfection, FACS analysis was conducted to monitortransfection efficiency. Only experiments with transfectionefficiency>80% were analyzed further. Cells were scrutinized by laserconfocal microscopy 4 hours after transfection of fl-siRNA to assurethat the siRNA was inside, rather than on the surface of, the cells atthat time point. Cell transfected with fl-siRNA exhibited a diffuseintracellular signal, rather than a surface pattern. Several siRNAs wereassessed and two were found to be effective in experiments with adequatetransfection efficiency (>90%). Real-Time RT-PCR and western analysis ofcell lysates at 48 hours post-transfection demonstrated that siRNAknocked down S14 protein levels and mRNA levels (mRNA levels were˜10-25% of controls). Cyclophilin mRNA was employed as a referencesequence, and did not vary among treatments.

Having demonstrated effective reduction in S14 levels using siRNA, twoaspects of S14 action in breast cancer cells were assessed. The effectof siRNA on T47D viable cell accumulation over time was determined.Cells accumulated in the presence of the control siRNA, whereassignificant cell dropout was observed in the presence of the siRNA. Acomparable effect was observed with another S14-siRNA. It was alsodetermined whether a reduction in S14 expression would abrogate theinduction of FAS mRNA by progestin. Real-time RT-PCR analysis usingFAS-specific primers demonstrated a significant reduction in FAS messagein response to S14 siRNAs within 48 hours post-transfection.

The effect of adenovirus harboring S14 cDNA in the antisense orientationwas also examined to determine if the effect of siRNA on T47D cellgrowth would be observed when using this alternative technique. Growthof cells exposed to the control virus did not differ from the uninfectedgroup 120 hours after infection. Inspection showed that a major celldropout began in the Ad-S14-AS group 72-96 hours after infection, andthis was confirmed by MTS assay. Lipid synthetic rate of the cellsbefore the onset of apoptosis (48 and 72 hours post-infection,respectively) was also examined. Incorporation of labeled acetate intolipids was not different among the groups 48 hours post-infection,whereas a sharp reduction was seen in antisense-treated wells 24 hourslater. An in situ TUNEL assay 96 hours post-infection showed evidence ofapoptosis in the antisense-treated group. Apoptotic effects of Ad-S14-ASwere also seen in MCF7 and SKBR3 breast cancer cells, whereas theantisense adenovirus had no effect on the accumulation of HepG2 cells.

Analogous studies were performed using siRNA transfection in MCF10acells, a non-tumorigenic human mammary epithelial line that expresseslow levels of lipogenic enzymes and is not susceptible to killing by FASenzyme inhibition as are transformed MCF10a or breast cancer cell lines(Yang, et al. (2002) Exp. Cell Res. 279:80-90). FACS analysis 4 hoursafter transfection using fluorescent siRNA showed that MCF10a cells weretransfectable. T47D and MCF10a cell content of S14 and FAS mRNAs werecompared and levels of both messages were so low that some wells yieldedno signal. In contrast to T47D cells, MCF10a cell growth was notaffected by S14 siRNA. To further compare the phenotypes of the T47D andMCF10a cells, both cell lines were exposed to the fatty acid synthaseinhibitor cerulenin (10 μg/mL for 48 hours). This confirmed previousreports of the differential sensitivity of the lines to inhibition oflipogenesis (Pizer, et al. (1996) supra; Yang, et al. (2002) supra).

The data disclosed herein demonstrated FAS and S14 gene induction byprogestin in breast cancer cells (Joyeux, et al. (1989) Mol. Endocrinol.4:681-686; Heemers, et al. (2000) supra). Similarly SREBP-1c mRNA wasincreased by the steroid as well. SREBP-1c gene expression has beenshown to be inducible in other circumstances, including stimulation byinsulin and high glucose in rat hepatocytes (Foretz, et al. (1999)supra), by refeeding in mouse liver and adipocytes (Kim, et al. (1998)J. Clin. Invest. 101:1-9), and by androgen in prostate cancer cells(Heemers, et al. (2001) supra). States of increased SREBP-1c actionresult in enhanced turnover of the extranuclear precursor (Yabe, et al.(2003) Proc. Natl. Acad. Sci. USA 100:3155-3160), and augmentedproduction is required to maintain an activated steady-state. Inhibitionof SREBP-1c was found to reduce the capacity of progestin to enhance S14and FAS mRNA expression, thus providing direct evidence for itsrequirement in the action of the hormone in breast cancer cells.Abrogation of progestin-induced FAS reporter gene activity in theconstruct lacking the sterol response element was consistent with this.Conversely, a constitutively active SREBP-1c mutant increased expressionof the endogenous S14 and FAS genes in T47D breast cancer cells in theabsence or presence of progestin. In the presence of hormone, strikingsuperinduction of both mRNAs was observed with concurrent SREBP-1cstimulation. Moreover, increased and reduced S14 protein expressioneffected concordant changes in the growth of breast cancer cells.Therefore S14 and associated components of the lipid synthetic pathwayin breast tumors are useful as therapeutic targets for the preventionand treatment of breast cancer.

Accordingly, the present invention is also a method for treating breastcancer in a subject having or at risk of having breast cancer. A subjecthaving or suspected of having breast cancer may exhibit one or more ofthe typical signs or symptoms associated with the disease including alump, an area of thickening, or a dimple in the breast or the lesscommon signs include breast swelling and redness or an enlarged underarmlymph node. Subjects at risk of having breast cancer include those witha family member or family history of having breast cancer or who haveinherited an abnormal breast cancer gene.

A subject having or at risk of having a breast cancer is administered aneffective amount of an agent that inhibits the expression or activity ofS14 (i.e., an S14 inhibitor) thereby inducing apoptosis in breast cancercells and having a beneficial or desired clinical result. Beneficial ordesired clinical results can include, but are not limited to,alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, preventing spread of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. Treatment can also mean prolonging survival as compared toexpected survival if not receiving treatment. As will be understood bythe skilled artisan, the signs or symptoms of the breast cancer can varywith the stage of the cancer and the signs or symptoms associated withvarious stages are well-known to the skilled clinician. See, forexample, The American Joint Committee on Cancer Staging Manual, SixthEdition.

An effective amount of an agent that inhibits the expression or activityof S14 is an amount sufficient to induce apoptosis in breast cancercells and effect beneficial or desired results, including clinicalresults. A reduction or decrease in S14 expression or activity isintended to mean a 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%decrease in expression or activity when compared to otherwise sameconditions wherein an S14 inhibitor is not present. As a result of sucha decrease in S14 expression or activity, apoptosis is induced in breastcancer cells of the subject thereby preventing, eliminating, stabilizingor alleviating one or more signs or symptoms of a breast cancer. Such adecrease in the expression or activity of S14 will be dependent on thesubject and the condition of the subject, the mode of administration ofthe agent, the stage of the cancer being prevented or treated and theparticular agent being employed.

As demonstrated herein, S14 inhibitors promote apoptotic cell death.Accordingly, the present invention also relates to a method for usingS14 inhibitors to modulate the growth of a breast cancer cell either invitro or in vivo. The method involves contacting a breast cancer cellwith an effective amount of an S14 inhibitor (i.e., an agent thatinhibits the expression or activity of S14) so that apoptosis of saidcell is induced or promoted. Induction or promotion of apoptosis of abreast cancer cell is intended to mean a 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98%, or 100% increase in cell death when compared to otherwisesame conditions wherein the S14 inhibitor is not present. Measurementsof cell death can be performed using methods well-known to those ofskill in the art, e.g., measuring a reduction in tumor size, an decreasein cell number or the expression of apoptosis marker proteins (e.g.,caspases).

In particular embodiments, the S14 inhibitor is an siRNA (e.g.,targeting nucleic acids set forth in SEQ ID NO:4 or SEQ ID NO:5) or anantisense molecule (e.g., an antisense molecule of a nucleic acidencoding a protein set forth in SEQ ID NO:1).

For use in accordance with the present invention, an S14 inhibitor cangenerally be formulated into a pharmaceutical composition foradministration to human subjects in a biologically compatible formsuitable for administration in vivo. A pharmaceutical compositioncontaining an S14 inhibitor can be prepared by known methods for thepreparation of pharmaceutically acceptable compositions which can beadministered to subjects, such that an effective amount of the activesubstance is combined in a mixture with a pharmaceutically acceptablevehicle. Generally, a pharmaceutical composition contains, for example,0.1 to 99.5%, or more suitably 0.5 to 90%, of active ingredient incombination with a pharmaceutically acceptable vehicle. Suitablevehicles are described, for example, in Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippincottWilliams & Wilkins: Philadelphia, Pa., 2000. Such compositions include,albeit not exclusively, solutions of an S14 inhibitor in associationwith one or more pharmaceutically acceptable vehicles or diluents, andcontained in buffered solutions with a suitable pH and iso-osmotic withthe physiological fluids.

An S14 inhibitor can be used in accordance with the method of thepresent invention in the form of a salt, solvate or as hydrate. Allforms are within the scope of the invention.

In accordance with the methods of the invention, an S14 inhibitor can beadministered to a subject in a variety of forms depending on theselected route of administration, as will be understood by those skilledin the art. An S14 inhibitor can be administered, for example, by oral,parenteral, buccal, sublingual, nasal, rectal, patch, pump ortransdermal administration and the pharmaceutical compositionsformulated accordingly. Parenteral administration includes intravenous,intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal,intrapulmonary, intrathecal, rectal and topical modes of administration.Parenteral administration can be by continuous infusion over a selectedperiod of time.

An S14 inhibitor can be orally administered, for example, with an inertdiluent or with an assimilable edible carder, or it can be enclosed inhard or soft shell gelatin capsules, or it can be compressed intotablets, or it can be incorporated directly with the food of the diet.For oral therapeutic administration, an S14 inhibitor can beincorporated with an excipient and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like.

An S14 inhibitor can also be administered parenterally. Solutions of anS14 inhibitor can be prepared in water suitably mixed with a surfactantsuch as hydroxypropylcellulose. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, DMSO and mixtures thereof with orwithout alcohol, and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms. A person skilled in the art would know how to preparesuitable formulations using conventional procedures and ingredients forthe selection and preparation of suitable formulations.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersion and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In all casesthe form must be sterile and must be fluid to the extent that easysyringability exists.

Compositions for nasal administration can conveniently be formulated asaerosols, drops, gels and powders. Aerosol formulations typicallycomprise a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container can bea unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant which can be a compressed gassuch as compressed air or an organic propellant such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, wherein the active ingredient isformulated with a carrier such as sugar, acacia, tragacanth, or gelatinand glycerine.

An S14 inhibitor can be administered to a subject alone or incombination with pharmaceutically acceptable carriers, as noted above,the proportion of which is determined by the solubility and chemicalnature of the inhibitory agent, chosen route of administration andstandard pharmaceutical practice.

The dosage of an S14 inhibitor can vary depending on many factors suchas the mode of administration, the age, health and weight of therecipient, the nature and extent of the symptoms, the frequency of thetreatment and the type of concurrent treatment, if any and the clearancerate of the compound in the subject to be treated. One of skill in theart can determine the appropriate dosage based on the above factors. AnS14 inhibitor can be administered initially in a suitable dosage thatcan be adjusted as required, depending on the clinical response.

Actual dosage levels and time course of administration of an S14inhibitor in a pharmaceutical composition can vary so as to obtain anamount of an S14 inhibitor which is effective to achieve the desiredtherapeutic response for a particular subject and mode ofadministration, without being toxic to the subject.

An S14 inhibitor can be used alone or in combination with a secondanticancer agent. For example, an S14 inhibitor can be administered incombination with calcitriol or other vitamin D receptor agonists. Othertreatments which can be combined with the use of an S14 inhibitorinclude, but are not limited to, Doxorubicin, Paclitaxel, Methotrexate,5-Fluorouracil, Docetaxel, Thiotepa, Cisplatin, Estrogen receptormodulators such as Tamoxifen and Toremifene, Estrogens (e.g.,diethylstilbestrol), Androgens (e.g., fluoxymesterone),Gonadotropin-Releasing Hormone (GnRH), Anastrozole, Aromatase inhibitors(antineoplastics), Vinorelbine tartrate, Gemcitabine hydrochloride,Progestins (e.g., Medroxyprogesterone acetate, Megestrole acetate),Trastuzumab (HERCEPTIN®), or Cyclophosphamide.

An S14 inhibitor can be administered prior to the administration of thesecond agent, simultaneously with the second agent, or after theadministration of the second agent. Furthermore, an S14 inhibitor can beadministered in a proform which is converted into its active metabolite,or more active metabolite in vivo.

As indicated, a S14 inhibitor is intended to include an agent thatinhibits the activity or expression of S14. Such agents include thosedisclosed herein as well as other agents identified in screening assaysfor agents that inhibit the expression or activity of S14. Agents of theinvention can be ligands or antibodies that bind to S14, agents thatprevent nuclear localization of S14, agents that block multimerizationof S14, or agents that block transcription or replication of S14 DNA ortranslation of S14 mRNA. Agents can be proteins or fragments thereof,small molecules, or nucleic acid molecules.

Various commercial sources are well-known in the art for providing smallmolecule libraries for the identification of useful compounds. Screeningof such libraries of test compounds, including combinatorially generatedlibraries (e.g., peptide libraries), is a rapid and efficient way toscreen large numbers of related (and unrelated) compounds for S14inhibitory activity. Combinatorial approaches also lend themselves torapid evolution of potential drugs by the creation of second, third andfourth generation compounds modeled of active, but otherwise undesirablecompounds.

Test agents can include fragments or parts of naturally-occurringcompounds, or may be found as active combinations of known compounds,which are otherwise inactive. It is contemplated that compounds isolatedfrom natural sources, such as animals, bacteria, fungi, plant sources,including leaves and bark, and marine samples can be assayed as testagents for the presence of potentially useful pharmaceutical agents. Itwill be understood that the test agents to be screened could also bederived or synthesized from chemical compositions or man-made compounds.Thus, it is understood that the test agent identified by the presentinvention can be peptide, polypeptide, polynucleotide, small moleculeinhibitors or any other compounds that can be screened for or designedthrough rational drug design.

Other suitable S14 inhibitors include siRNA molecules, antisensemolecules, ribozymes, and antibodies (including single chainantibodies), each of which would be specific for the target S14molecule. Examples of such agents are described herein. For example, anantisense molecule that binds to a translational or transcriptionalstart site, or splice junction, would be an ideal S14 inhibitors.

An inhibitor according to the present invention can be one which exertsits inhibitory effect upstream, downstream or directly on S14 nucleicacid or protein. Regardless of the type of inhibitor identified by thepresent screening methods, the effect of the inhibition by such an agentresults in reduction in the expression or activity of S14 as compared tothat observed in the absence of the added inhibitor agent.

An inexpensive and easy assay for identifying S14 inhibitors is an invitro assay. Such assays generally use isolated molecules, can be runquickly and in large numbers, thereby increasing the amount ofinformation obtainable in a short period of time. A variety of vesselscan be used to run the assays, including test tubes, plates, dishes andother surfaces such as dipsticks or beads.

One example of a cell-free assay is a binding assay. While not directlyaddressing function, the ability of an inhibitor to bind to a targetmolecule in a specific fashion is strong evidence of a relatedbiological effect. For example, binding of a molecule to a target can,in and of itself, be inhibitory, due to steric, allosteric orcharge-charge interactions. The target can be either free in solution,fixed to a support, expressed in or on the surface of a cell. Either thetarget or the compound can be labeled, thereby permitting determining ofbinding. Usually, the target will be the labeled species, decreasing thechance that the labeling will interfere with or enhance binding.Competitive binding formats can be performed in which one of the agentsis labeled, and one can measure the amount of free label versus boundlabel to determine the effect on binding.

A technique for high throughput screening of compounds is described inWO 84/03564. Large numbers of small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. Bound polypeptide is detected by various methods.

In a yeast two-hybrid assay using S14 as bait (Cunningham, et al. (1997)Endocrinol. 138:5184-5188), approximately 100 strongly interactingclones were identified, all of which were S14 cDNAs. This analysisindicated that S14 formed multimers. The relevance of the S14-selfinteraction in the yeast two-hybrid system to mammalian cells wasverified by co-transfecting S14 constructs bearing two different tags,and demonstrating that HA-tagged S14 co-purified with GST-S14, but notGST, on glutathione affinity chromatography (Cunningham (1997) supra).Analysis of deletion mutants in yeast indicated that the C-terminal“zipper domain” of S14, which is predicted to be alpha-helical, wasrequired for multimer assembly. In addition, screening a cDNA librarydevoid of S14 (HeLa cell, as assessed by RT-PCR), screening with S14bait lacking the zipper domain, and digesting the cDNA library with arare-cutting restriction enzyme that cleaves the S14 sequence did notyield additional binding partners.

Analytical equilibrium ultracentrifugation and light-scatteringindicated that S14 forms a homotetramer in solution. Secondary structureprediction software (Jpred) identified 20 residues in S14 domain IIIwith the potential to form a coiled-coil, typical of an oligomerizationmotif. Circular dichroism experiments using a 20-mer representingresidues 120-139 of that domain showed a peak at 190, and a nadir at 210nM, typical of an α-helix, in 50% trifluoroethanol (i.e., Cottoneffect). In contrast to complete S14, the 20-mer was small enough forNMR spectroscopy. NMR data verified the helical configuration, anddisclosed that the helices interact in parallel. Modeling as a fourparallel helix bundle predicted several residues, particularly Tyr138 ofSEQ ID NO:1, to be key for stabilizing the tetrameric interface. ATyr138Ala S14 mutant was expressed in bacteria and, in contrast towild-type S14, was not found in the soluble fraction. Western blotanalysis confirmed that the mutant was confined to the insoluble cellpellet, indicating that Tyr138 is a crucial residue for assembly ofsoluble S14 tetramers.

These data demonstrate that S14 forms multimers in vitro and in vivo,and that it interacts with 140 and 10 kD peptides in breast cancercells. Domain III is demonstrated to be helical in solution, where itforms a tetramer in parallel orientation. Moreover, within Domain IIITyr138 is a vulnerable point in assembly of tetrameric S14. Of note,Tyr138 is conserved in S14 and S14-RP homologs from zebrafish to humans(Wang, et al. (2004) Gene 332:79-88).

Accordingly, particular embodiments of the present invention embrace invitro assays for identifying a test agent that decreases themultimerization of S14 thereby blocking activity of S14. In accordancewith such an assay, S14 is contacted with a test agent andmultimerization is determined, e.g., by detecting soluble multimers ofS14 via western blot analysis. Agents that block multimerization of S14are useful for inducing apoptosis in a breast cancer cell and in thetreatment of breast cancer.

Moreover, it is contemplated that given the importance of Tyr138 formultimerization, the step of determining the multimerization of Spot 14can be carried out by computer modeling using docking programs such asGRAM, DOCK, or AUTODOCK to identify agents which interact with Tyr138thereby interfering with multimerization. Computer programs can beemployed to estimate the attraction, repulsion, and steric hindrance ofa compound to Tyr138. Generally the tighter the fit (e.g., the lower thesteric hindrance, and/or the greater the attractive force) the morepotent the potential drug will be since these properties are consistentwith a tighter binding constant. Furthermore, the more specificity inthe design of a potential drug the more likely that the drug will notinterfere with other proteins thereby minimizing potential side-effects.

The present invention also contemplates the screening of compounds fortheir ability to inhibit S14 expression or activity in cells. Variouscell lines can be utilized for such screening assays, including cellsspecifically engineered for this purpose. For example, cells containingS14 regulatory elements (e.g., promoter) operatively linked to areporter molecule are specifically contemplated for use in identifyingagents that inhibit the expression of S14. Depending on the assay, cellscan be examined for different physiologic phenotypes (e.g., induction ofapoptosis) or, alternatively, molecular analysis can be performed, forexample, looking at protein expression, mRNA expression (includingdifferential display of whole cell or polyA RNA) and others.

In vivo assays can involve the use of various cell or animal models,including transgenic animals that have been engineered, e.g., to carrymarkers that can be used to measure the ability of a test agent toeffect the expression or activity of S14. In such assays, one or moretest agents are contacted with a cell, and the ability of the testagent(s) to inhibit the expression or activity of S14, as compared to asimilar cell not treated with the test agent(s), is determined.Determination can be achieved by detecting the binding of ligands orantibodies to S14, detecting nuclear localization of S14, detectingmultimerization of S14, or detecting transcription or replication of S14DNA or translation of S14 mRNA.

For example, agents that inhibit the transcription or translation of S14can be identified by employing nucleic acids encoding a reporter (e.g.,GFP or luciferase) operably linked to the S14 promoter (and 5′ and/or 3′untranslated regions; see Campbell, et al. (2003) supra), wherein thepresence of the reporter in a cell is indicative of the transcriptionand translation of S14. As such, an agent that decreases the presence orlevel of the reporter in the test cell when compared to a test cell notcontacted with the agent, indicates that the agent decreases thepresence or level of S14 and is therefore useful for inducing orpromoting apoptosis in breast cancer cells. Such cell-based reporterassays are routinely carried out by the skilled artisan with more detaildiscussed in Zysk (1998) Comb. Chem. High Throughput Screen 1(4):171-83and Gonzalez et al. (1998) Curr. Opin. Biotechnol. 9(6):624-31.

Agents identified in accordance with the methods disclosed herein areuseful in inducing apoptosis in breast cancer cells and in the treatmentof breast cancer.

The following non-limiting examples are presented to further illustratethe invention.

EXAMPLES Example 1 Patient Population

From a database of 700 patients with blinded demographic information ontumor characteristics, including size, histological grade, lymph nodestatus, and expression of conventional tumor markers (e.g., estrogenreceptor, progesterone receptor, and Her2/neu) was selected 132 tumors.Of the selected tumors, 44 were consecutive cases of DCIS, 34 wereconsecutive cases of node (−) breast cancer, 54 were consecutive casesof node (+) breast cancer and 20 were mammary gland samples from womenundergoing reduction mammoplasty who had no history of breast cancer (10pre- and 10 post-menopausal).

Example 2 Determination of Conventional Tumor Markers

Immunostaining of tumor cell nuclei for estrogen receptor andprogesterone receptor expression was defined as “negative” (noimmunostaining); “equivocal” (1-15% tumor cell nuclei staining); and“positive” (>15% tumor cell nuclei staining). C-erb-B2 surface proteinexpression was scored immunohistochemically as 0 through 3+, accordingto adapted criteria defined in the HERCEPTEST. Score 0 was defined asabsent or faint membranous immunostaining in <33% of the tumor cells.Score 1+ was defined as barely perceptible partial membranous stainingin >33% of the tumor cells. Score 2+ was defined as weak to moderatestaining of the entire cell membrane in >33% of the tumor cells. Score3+ was defined as strong staining of the entire cell membrane in >90% ofthe tumor cells. Scores of 0, 1+ and 2+ were deemed as negative, andscores of 3+ to indicate overexpression. Her2neu slides were read by oneexperienced pathologist and the rate of cases scored as 2+ thatexhibited a positive signal by fluorescent in situ hybridization (FISH)was 2.5%. In view of cost of that test and the excellent reproducibilityof immunohistochemical analysis for this antigen, cases graded as 2+ byFISH were not routinely analyzed.

Example 3 Anti-Human S14 Antibody Production

A monoclonal antibody selected for specifically binding to a S14 proteinof SEQ ID NO:1 in breast tissue or cells was prepared by generatinghybridomas from splenocytes of mice immunized withglutathione-S-transferase (GST)-tagged human S14 expressed from vectorpGEX-3× (Amersham, Piscataway, N.J.) in E. coli. Female Balb/C mice wereimmunized intraperitoneally with 50 μg GST-S14 mixed in RIBI adjuvant(Sigma, St. Louis, Mo.), and boosted with 20 μg antigen in adjuvant 3and 6 weeks later. Splenocytes were fused with NS1 cells (ATCC,Manassas, Va.) days later. Screening was by ELISA using wells coatedwith His6-tagged S14 expressed in bacteria from vector PROEX-HT (LifeTechnologies, Gaithersburg, Md.). Crude supernatant (1:1000 dilution ofhybridoma “KVB7”, an IG2a) or anti-HA (Sigma, St. Louis, Mo.) were usedin western analysis with protein A-alkaline phosphatase conjugate fordetection of S14 or HA-tagged S14-related peptide, respectively, asdescribed (Kinlaw, et al. (1992) supra).

Example 4 Immunohistochemistry

Tissues were fixed in 10% buffered formalin (Biochemical Science Inc,Swedesboro, N.J.), dehydrated through graded alcohols, and paraffinembedded. Tissue sections (4 μm) were coated with adhesive (STA-ON;Surgipath Medical Industries, Inc, Richmond Ill.), mounted on glassslides and stained with hematoxylin for initial review. Estrogenreceptor protein and progesterone receptor protein expression (1:10 and1:40, respectively; Biogenix, San Ramon, Calif. with Citra Plus antigenretrieval) and C-erb-B2 surface protein expression (1:20; Biogenix, SanRamon, Calif. with Citra antigen retrieval) were assessedimmunohistochemically in the clinical laboratory at the time ofdiagnosis. S14 was detected with crude supernatant from a hybridomadesignated K/IIIC5.1, which produces anti-S14 antibody, an IG2a. FAS wasdetected with an affinity-purified rabbit anti-human FAS IgGpreparation, (Immuno-Biological Laboratories Co., Gunma, Japan) 1:100 at3 μg/mL. Cyclin D1 was detected with the mouse monoclonal, 1:100(Biocare, Walnut Creek, Calif.) with Borg antigen retrieval according topublished data (Petty, et al. (2004) supra). Tissue sections were cut,deparaffinized with xylene and hydrated through graded alcohols.Sections were mounted on Biogenix Plus slides (San Ramon, Calif.) andepitope retrieval was carried out in a steamer under pressure, usingCitra, Citra Plus or Borg antigen retrieval buffers (Biogenix, SanRamon, Calif.). Slides were rinsed in distilled water, soaked inphosphate-buffered saline and immunostained in a BioGenix I-6000autostainer (San Ramon, Calif.) using the Biotin-Streptavidin Amplifiedsystem, with identical timing of incubations and washes for all cases.Diaminobenzidine was applied for visualization. Slides werecounterstained with hematoxylin, dehydrated through graded alcohols andcoverslipped with Richard Allen mounting medium (Richard Allen Medical,Richland, Mich.).

Example 5 Determination of S14, FAS and Cyclin D1 Levels

All immunoslides were semi-quantitatively scored by one Pathologist. Foreach antibody, 20 randomly chosen cases were reviewed by a secondpathologist to confirm immunoscoring reproducibility. S14 and FAS werescored 0 through 2+ according to the intensity of immunostaining(nuclear for S14, cytoplasmic for FAS). Semi-quantitive analysis for thedetermination of marker levels by immunohistochemical analysis iswell-known in the art. See, for example, Petty, et al. (2004) Clin.Canc. Res. 10:7547-7554. In accordance with established methods, score 0was defined as no immunostaining; score 1+ was defined as weak, diffuseimmunostaining; score 2+ was defined as strong, diffuse immunostaining.Cyclin D1 was scored 0 through 3+ according to the percentage of tumornuclei staining (irrespective of the intensity) using published data(Petty, et al. (2004) supra) as follows: score 0 was defined as noimmunostaining; score 1+ was defined as <25% of tumor cell nucleistaining; score 2+ as 25-75% of tumor cell nuclei staining; score 3+as >75% of tumor cell nuclei staining.

Example 6 Statistical Analyses of Clinical Data

Confidence intervals for rates were calculated using exact binomialmethods. Comparisons for S14 and cyclin D1 overexpression between groupswere performed using Fisher's exact test. Time to disease recurrencebetween groups was compared using Kaplan-Meier survival estimates andthe log-rank test. Proportional hazards regression was also used tojointly examine the influence of the stage, prognostic factors, and S14overexpression on the time to recurrence.

Example 7 S14 as a Prognostic Marker for Breast Cancer

To demonstrate that S14 is a marker for breast cancer, the presence ofS14 in breast tissues was initially analyzed using a commerciallyavailable antibody preparation to S14; however, this antibodypreparation was not sufficient for detecting differential expression ofS14 in breast tissue. Accordingly, a monoclonal antibody selected forspecifically binding to S14 in breast tissue was generated. To analyzethe specificity of the S14 monoclonal antibody, western blot analysis ofT47D human breast cancer cells was performed. To increase expression ofS14 in these cells, either a progestin (10 nM R5020) (Heemers, et al.(2000) Biochem. Biophys. Res. Commun. 269:209-212) oradenovirally-delivered sterol response element-binding protein-1c(SREBP-1), a transcription factor that induces the expression of genesconcerned with lipogenesis (Rawson (2003) Nat. Rev. Mol. Cell Biol.4:631-6) was applied as a stimulus. To decrease expression of S14, T47Dhuman breast cancer cells were exposed to an S14 short inhibitory RNA.In each instance, a single S14 band of appropriate mobility (˜16 kD) wasobserved, the intensity of which was concordant with the level of S14mRNA.

Resting and lactating human mammary gland were also examined for S14expression using the S14-specific monoclonal antibody inimmunohistochemistry; lactation is a major stimulus for S14 expressionin epithelium of the lobuloalveolar units (Moncur, et al. (1998) supra).Resting mammary showed primarily white adipocytes of the fat pad withstrong nuclear and some cytoplasmic staining, whereas the lactiferousduct epithelium was essentially devoid of S14. Lactating mammary glandshowed strong staining in nuclei and cytoplasm of the epithelial cells,whereas staining for FAS showed an intense cytoplasmic signal, withsparing of the nuclei.

Further, immunohistochemical analysis of breast cancer was conductedusing the S14-specific monoclonal antibody. Both ductal carcinoma insitu (DCIS), a form of non-invasive breast cancer, and invasive breastcancer tissues were stained for S14 and FAS. As observed in rat liver(Kinlaw, et al. (1989) J. Biol. Chem. 264:19779-19783) and breast cancer(Moncur, et al. (1998) supra) using polyclonal IgG preparations inimmunohistochemistry, the S14-specific antibody primarily stained thenucleus. FAS immunoreactivity was cytosolic, as expected (Jensen, et al.(1995) J. Pathol. 176:343-352). Tumors of either type yielded no signalwhen primary antibodies were omitted.

Having established the presence of both S14 and FAS in breast cancertissue, the frequency of S14 and FAS expression in normal and malignantmammary tissue was examined. Of the 132 cases analyzed, S14 and FAS weredetectable in essentially all examples of normal mammary gland, DCIS,and invasive breast cancer (Table 1). The frequency of maximalexpression did not vary between DCIS and invasive cancers, and thescores did not differ between invasive cancers with (34 cases) and thosewithout (54 cases) lymph node metastases.

TABLE 1 S14 Staining FAS Staining Tissue (n) (%) (%) DCIS (44)Detectable 97 97 Maximal 68 97 Invasive breast cancer (88) Detectable 9999 Maximal 76 97 Normal mammary (20) Detectable 100 100 Maximal 60 70The detectable category for S14 or FAS refers to an immunohistochemicalscore of 1 or 2; the maximal category corresponds to scores of 2. Thenormal mammary gland group included samples from 10 pre- and 10post-menopausal women. The data were pooled because there was nosignificant difference in staining intensities between them.

The fraction of cases exhibiting maximal staining for S14, but not FAS,was significantly correlated with tumor grade and size (FIG. 1). S14expression in grade 1 DCIS was as likely to be maximal as it was innormal mammary epithelium (FIG. 1, Panel A), but the prevalence ofstrong staining increased with advancing grade, to 97% in grade 3 cases(p=0.003). In invasive cancers (FIG. 1, Panel B) a similar relationshipwas found, with 100% of grade 3 tumors (pooled lymph node negative andpositive) exhibiting a maximal signal (p<0.001). Invasive cancers alsoexhibited increased S14 expression as a function of tumor size (FIG. 1,Panel C). Strong staining was found in 58% of tumors<1.0 cm in size, andincreased to 80% and 83% for lesions 1.0-2.0 and >2.0 cm in diameter,respectively (p=0.05). The number of cases at each S14 score precludedmultivariate analysis, but there did appear to be an effect of tumorgrade independent of the S14 score. Among strongly staining-cases, noneof 11 grade I invasive tumors recurred, 2/19 grade 2 tumors recurred,and 12/26 grade 3 tumors recurred.

In DCIS, the relationship between FAS expression and tumor grade nearlyreached significance, with grade 1/2/3 cases exhibiting maximalexpression in 86/96/100% of cases, respectively (p=0.076). This isconsistent with a previous report (Milgraum, et al. (1997) Clin. Canc.Res. 3:2115-2120). No such relationship was observed in invasive cases,because all samples exhibited maximal expression of this antigen. Aswith S14, FAS content in the tumor correlated with invasive tumor size.Strong staining was found in 88% of tumors<1.0 cm in size, and increasedto 100% for 1.0-2.0 or >2.0 cm lesions (p=0.036).

In a comparison of the expression of S14, FAS, and conventional tumormarkers, there was no significant correlation between the expression ofS14 and estrogen receptor or progesterone receptor status in either DCISor invasive cases. Likewise, there was no association of a positivescore for Her2/neu with the S14 or FAS scores. However, Her2/neuamplification did adversely affect disease-free survival (p=0.046).

In both DCIS and invasive breast cancer there was no observedrelationship between the S14 score and the level of cyclin D1 expression(p=1.00 and 0.28, respectively), an art-recognized mammary oncogene(Hinds, et al. (1994) Proc. Natl. Acad. Sci. USA 91:709-713). Cyclin D1staining was significantly enhanced in DCIS and invasive cancer comparedto normal mammary epithelium, but did not correlate with tumor size ateither stage (p=0.42 and p=0.25, respectively). Likewise, Cyclin D1staining did not correlate with tumor grade in either DCIS or invasivedisease. In DCIS, strong staining for cyclin D1 was associated withestrogen receptor expression (p=0.028). The association of cyclin D1expression and that of progesterone receptor was also statisticallysignificant (p=0.05). In invasive cases, the association betweenprogesterone receptor and strong cyclin D1 expression nearly reachedsignificance (p=0.058). However, no association was found between thecyclin D1 score and disease-free survival (p=0.4).

Kaplan-Meier analysis of recurrence after primary treatment of initiallyinvasive tumors, with or without lymph node involvement at the time ofsurgery, revealed a significant relationship (p=0.015) between the levelof S14 expression in the primary tumor and disease-free survival overthe ensuing 3000 days (FIG. 2). No cases exhibiting a score of 0 or 1+(n=21) recurred, whereas 32% of tumors with maximal S14 content recurred(n=67).

Separable effects of S14 and the presence of nodal metastases at initialsurgery were observed on disease-free survival in invasive disease (FIG.3). Among 34 node-negative cases, only one recurrence was seen, and itwas among the 23 cases with strong S14 expression. Among node-positivecases with weak S14 expression, no recurrence was observed on follow-up.In contrast, of the node positive cases with strong staining, 14 (32%)developed recurrent disease (p<0.0001).

Example 8 Recombinant Adenovirus

Adenovirus harboring dominant-negative and constitutively activeSREBP-1c mutants are known in the art (Foretz, et al. (1999) supra).β-Galactosidase gene (negative control; Ad-β-gal) and full-length ratS14 cDNA (Kinlaw, et al. (1992) Endocrinol. 131:3120-3122) in both thesense orientation (Ad-S14) and antisense orientation (Ad-S14-AS) wereinserted into adenoviral DNA (CLONTECH, Mountain View, Calif.) asdescribed (Mizuguchi, et al. (1998) Human Gene Therapy 9:2577-2583).Viruses were propagated in HEK293 cells (ATCC, Manassas, Va.) andtitered by immunocytochemical analysis of capsid protein (Rapid-Titer;CLONTECH, Mountain View, Calif.). The multiplicity of infection requiredfor quantitative infection was determined by staining Ad-β-gal-infectedwells.

Example 9 Cell Culture and Infection

T47D cells (ATCC, Manassas, Va.) were grown in RPMI 1640 plus 10 μg/mLinsulin, HEK293 cells (ATCC, Manassas, Va.) in RPMI 1640, and MCF10acells in DMEM/F12 plus 4 mg/mL insulin, 20 μg/mL epidermal growthfactor, and mg/mL hydrocortisone. Media contained penicillin,streptomycin, 4 mM glutamine, 25 mM glucose unless noted otherwise, and10% fetal calf serum unless noted. Charcoal-stripped fetal calf serum(HYCLONE, Logan, Utah) was used in studies involving R5020 or R1881 (10nM, New England Nuclear); an equal volume of ethanol vehicle was addedto control cultures. Cerulenin (SIGMA, St. Louis, Mo.) was used at 10μg/mL.

Example 10 Plasmid Transfection

T47D or HEK 293 cells were plated at 50% confluence in 75 cm² flasks andthe next morning were transfected with 8 μg plasmid DNA in 48 μL FUGENE(Roche, Indianapolis, Ind.) in 5% charcoal-stripped serum-containingmedia without antibiotics. To ensure uniform transfection efficiency,cells were trypsinized, mixed, and redistributed into 6-well plates 8hours later. Forty-eight hours post-transfection, culture medium wasremoved, and extracts prepared in reporter lysis buffer (250 μL/well;PROMEGA, Madison, Wis.). Lysates (20 μL) were assessed for luciferaseactivity using an LMaxII384 luminometer (MOLECULAR DEVICES, Sunnyvale,Calif.), and normalized to protein concentrations (BCA Protein Assay;Pierce, Rockland, Ill.).

Example 11 Transfection of siRNA

Cells were plated at ˜70% confluency in 60-mm dishes the day beforetransfection with 20 μg siRNA in 333 μL diluent supplied by QIAGEN, and120 μL of RNAIFECT Transfection Reagent (QIAGEN, Valencia, Calif.). ThesiRNAs (Dharmacon, Chicago, Ill.) targeted the following sequences inS14 mRNA: siRNA#1, 5′-gga aat gac ggg aca agt t-3′ (SEQ ID NO:4); andsiRNA#2, 5′-cag ccg agg tgc aca aca t-3′ (SEQ ID NO:5). Scrambled siRNAswere employed as controls. Complexes were incubated at room temperaturefor 15 minutes and added drop-wise to cultures. After 24 hours, cellswere trypsinized and redistributed into 4 wells of a 12-well plate toensure uniform transfection efficiency, in media containing hormone orvehicle.

Example 12 Reverse Transcriptase-PCR

Total RNA (500 ng) harvested in TRIZOL™ was used as template with theGIBCO/BRL “OneStep” kit. Primers (forward/reverse) for cyclophilin were5′-gga tgg caa gca tgt ggt g-3′ (SEQ ID NO:6) and 5′-tgt cca cag tca gcaatg g-3′ (SEQ ID NO:7); S14, 5′-cca tct gtg tgg atg tgg acc-3′ (SEQ IDNO:8) and 5′-agc atc ccg gag aac tga gcc-3′ (SEQ ID NO:9); SREBP-1a,5′-tca gcg agg cgg ctt tgg agc ag-3′ (SEQ ID NO:10) and 5′-cat gtc ttcgat gtc ggt cag-3′ (SEQ ID NO:11; Shimomura, et al. (1997) supra);SREBP-1c, 5′-gga ggg gta ggg cca acg gcc t-3′ (SEQ ID NO:12) and 5′-catgtc ttc gaa agt gca atc c-3′ (SEQ ID NO:13; Shimomura, et al. (1997)supra); and FAS, 5′-aca ggg aca acc tgg agt tct-3′ (SEQ ID NO:14) and5′-ctg tgg tcc cac ttg atg agt-3′ (SEQ ID NO:15; Field, et al. (2001) J.Lipid Res. 42:1687-1698). S14-RP was analyzed with two sets of primers,one that amplified the entire coding region (5′-acc cgg ccg acc atccc-3′ (SEQ ID NO:16) and 5′-agt ttg cag tct gcc ctt ccc-3′ (SEQ IDNO:17)) and a nested pair (5′-ccg ggt tag aca acg atg tt-3′ (SEQ IDNO:18) and 5′-tgg ctg tac atg tcc cga gag-3′ (SEQ ID NO:19)). PGC-1βprimers were 5′-acc tca cct cgg cac agt get-3′ (SEQ ID NO:20) and 5′-tcaccc ggc tcc ttg tcc t-3′ (SEQ ID NO:21), and those for CHREBP were5′-ccg cct gag gat gcc tac gtc-3′ (SEQ ID NO:22) and 5′-gga ggc ggg agttgg taa aga-3′ (SEQ ID NO:23). Sizes of the products in by were: S14365; SREBP-1a 80; SREBP-1c 80; FAS 159; S14-RP 724 and 132; PGC-1β 99,and CHREBP 116. Amplification was at 55° C. for 30 minutes, 94° C. for 2minutes, followed by 15 cycles each of 94° C. for 30 seconds, 57° C. for30 seconds, 72° C. for 1 minute; 94° C. for 30 seconds, 62° C. for 30seconds, 72° C. for 1 minute; 94° C. for seconds, 55° C. for 30 seconds,72° C. for 1 minute, followed by extension at 72° C. for 2 minutes.Reactions using primers that did not span introns were alwaysaccompanied by a control PCR devoid of reverse transcriptase, whichnever yielded a product.

Example 13 Real-Time Reverse Transcriptase PCR

Total RNA was isolated using RNEASY minicolumns from cell extractsprepared with QIASHREDDER (QIAGEN, Valencia, Calif.). RNA integrity wasassessed by visualization on agarose gels, and contamination withgenomic DNA was excluded by failure to obtain a PCR product usingprimers for cyclophilin A. RNA (1 μg) was reverse-transcribed withM-MuLV reverse transcriptase and p(dt)18 (NEW ENGLAND BIOLABS, Ipswich,Mass.). Product (50 ng) was added to 96-well plates (in duplicate) withprimers and SYBR Green reaction mix (PE Biosystems). PCR (BIO-RAD MYIQICYCLER) commenced with heat activation for AMPLITAQ GOLD DNA polymerase(Roche, Indianapolis, Ind.) (95° C. for 10 minutes), followed bydenaturation (95° C. for 30 seconds), annealing (57° C. for seconds),extension (72° C. for 30 seconds), and data acquisition at the end ofthe extension step, for 40 cycles. Dilutions of cloned cDNA fragmentsfrom each mRNA assayed were included to provide a standard curve. MYIQOptical System Software (BIO-RAD, Waltham, Mass.) was used to assesssignals during the log-linear accumulation phase, calculated as ngtemplate per tube compared to the standard curve, which was linearacross 6 logs of input. Values were normalized to the signal obtainedfrom the same sample using primers for cyclophilin A and reported inarbitrary units. Melting curves assured that signals arose from singleproducts, and wells without template were included to survey forcontamination.

Example 14 Cell Growth

Cells (20,000/well) were seeded in 12-well plates in media containingstripped fetal calf serum. Medium was replaced after 12 hours, and 12hours later with media containing 10 nM R1881 (or R5020) or vehicle.Media were replaced again after 3 days, and growth was assayed on the6^(th) day after hormone addition. In experiments using adenovirus,cells (20,000/well) seeded in medium containing 10% fetal calf serumwere infected the following morning. Media were changed after 1 hour,and again 3 days later. Cell accumulation was measured by the3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay (PROMEGA, Madison, Wis.). Oxidation of MTS by viablemitochondria yields a product that absorbs at 490 nM. MTS data showed astrong correlation with DNA content/well under a variety of metaboliccircumstances.

Example 15 Lipid Synthesis

14-[C]-acetate (4 μCi/mL; Sigma, St. Louis, Mo.) was added to media for3 hours, and incorporation into lipid was determined as in (Kinlaw, etal. (1995) J. Biol. Chem. 270:16615-16618), using standard methods(Bligh & Dyer (1959) Can. J. Biochem. Physiol. 37:911-917)

Example 16 Statistical Analysis

All experiments were repeated at least once. Comparison of two groupswas by two-tailed Student's t-test. Comparisons between more than twogroups was by two-way analysis of variance (Wilkinson, et al. (1992)SYSTAT: Statistics, Version 5.2 Edition, p. 724, Evanston, Ill.: SYSTAT,Inc.).

Example 17 Mouse Model of Breast Cancer

To further determine the role of S14 in breast cancer, a conditionalknockout model is employed. It is contemplated that a completemammary-specific knockout will not be lethal, and that it will permitthe flexibility to analyze S14 function in selected mammary epithelialsubtypes or in pregnancy-dependent models using appropriateCre-expressing mice (Wagner et al. (2003) Mol. Cell. Biol. 23:150-162).As such, a mice with germline transmission of a floxed S14 allele hasbeen produced and will be used in conjunction with mice harboringtransgenes for both mammary epithelial Cre recombinase expression and amammary oncogene. In view of the nexus between Her2/neu signaling, S14,and the lipogenic breast cancer phenotype, the MMTV-Her2/neu GEM modelsuitable (Holland, ed. (2004) Mammary Gland Cancer, John Wiley).

What is claimed is:
 1. A method for inducing apoptosis in a breastcancer cell comprising contacting a breast cancer cell with an agentthat inhibits the expression or activity of Spot 14 thereby inducingapoptosis in the breast cancer cell, wherein said agent comprises ansiRNA targeting a nucleic acid sequence comprising SEQ ID NO:4 or SEQ IDNO:5 or an antisense molecule of a nucleic acid encoding a protein setforth in SEQ ID NO:1.
 2. A method for inhibiting growth of breast cancercells comprising administering to a subject with breast cancer an agentthat inhibits the expression or activity of Spot 14 in an amounteffective to inhibit growth of the breast cancer cells in the subject,wherein said agent comprises an siRNA targeting a nucleic acid sequencecomprising SEQ ID NO:4 or SEQ ID NO:5 or an antisense molecule of anucleic acid encoding a protein set forth in SEQ ID NO:1.
 3. A methodfor treating breast cancer comprising administering to a subject withbreast cancer an agent that inhibits the expression or activity of Spot14 in an amount effective to treat the breast cancer in the subject,wherein said agent comprises an siRNA targeting a nucleic acid sequencecomprising SEQ ID NO:4 or SEQ ID NO:5 or an antisense molecule of anucleic acid encoding a protein set forth in SEQ ID NO:1.